Various fermentation strategies have been used to produce proteins in sufficient quantities for laboratory, clinical or commercial use. Fed-batch fermentation has been used to provide increased proteins yields over those provided by simple batch fermentation methods. Fed-batch fermentation is a process in which, after an initial batch phase, a phase takes place in which one or more nutrients are supplied to the culture by feeding.
Generally, during the batch phase, cells are initially grown to a desired concentration. At this phase, cell growth is amplified and generally no target protein will be produced unless one adds an inducer, such as arabinose, lactose or isopropyl beta-D-thiogalactoside (IPTG), depending on the promoter, or there is some leakage of the promoter. During the feed phase, carbon source and other requirements are typically fed to a fermentor in a relatively concentrated liquid stream at a certain feed rate. Once a target cell density is achieved, a feed is commenced with the inducer or the inducer and other nutrients. In this phase, the emphasis is on protein production by the grown cells. Substrate (that is, the nutrients and the inducer) that is fed to the fermentor is at this stage used generally for cell growth and product synthesis. The cell growth is controlled by the feed rate to obtain an optimum cell growth and production of protein. During the protein production stage, an inducer must be added for recombinant organisms.
Protein expression on a medium comprising a common carbon source such as glucose or another sugar based carbon source and an inducer is satisfactory until limiting conditions arise at the end of the feed phase. Examples of limiting conditions include reduced oxygen concentration, reduced nutrients such as vitamins, carbon, nitrogen and accumulation of toxic compounds in the growth medium.
Fed-batch fermentation strategies often involve different forms of feedback control, including indirect and direct feedback to control the supply of nutrients. One such fed-batch fermentation method involves application of a feedback control algorithm by feeding nutrients in order to control a process parameter at a defined set point. For example, direct control of feed may be based on measurement of nutrient concentration. Feedback control is then directly related to cell activity throughout fermentation. Control parameters which have been used for feedback control of fermentations include pH value, on line measured cell density or dissolved oxygen tension (DOT).
However, the application of feedback algorithms is accompanied by a number of disadvantages. One such disadvantage is that the feed rate depends on current process parameters. Any disturbance to the process may affect the parameter thus distorting the feed rate and resulting protein yield. Such disadvantages are magnified as the process is scaled-up to produce increased protein quantities.
Another disadvantage of previously employed fed-batch strategies is that when using feed-back control, the specific growth rate cannot be exactly predefined or controlled, resulting in suboptimal yields in processes, where the product formation is dependent on growth.
Further, when carbon flux (for example, high glucose concentration) into the central metabolic pathway exceeds the maximum capacity of the Tricarboxylic Acid (TCA) cycle, by-products may accumulate. The accumulation of by-products could inhibit cell growth and protein production during fermentation.
Additionally, the various deficiencies of fed-batch fermentation methods often result in inefficient use of nutrient components. As such, the methods may be economically disadvantageous, particularly for large scale commercial protein production.
Previous approaches to recombinant protein expression through fed-batch fermentation, as described above, have various deficiencies. Given the importance of cost-effectively producing sufficient quantities of protein for various purposes, there is a need for an efficient fed-batch fermentation method that results in higher cell growth, increased product formation (that is, higher protein yield), and decreased by-product accumulation.